Dorina Avram

P300 FUNCTIONS AS A COACTIVATOR FOR THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA

The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor α in a ligand-enhanced manner. The PPARα-interacting domain of p300 was mapped to amino acids 39–117 which interacted strongly with PPARα but did not interact with retinoic acid receptor-γ or retinoid X receptor-α. Amino acids within the carboxyl terminus of PPARα as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARα and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARα and further characterize the molecular mechanisms of PPARα-mediated transcriptional regulation.

Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein.

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor α (PPARα), and PPARα ligands suppressed this interaction. In contrast to the interaction of PPARα with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARα ligand binding domain. NCoR was found to suppress PPARα-dependent transcriptional activation in the context of a PPARα·retinoid X receptor α (RXRα) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARα·RXRα heterodimeric complexes in mammalian cells. NCoR appears to influence PPARα signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Heterodimeric Interactions between Chicken Ovalbumin Upstream Promoter-Transcription Factor Family Members ARP1 and Ear2

Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.

Isolation and characterization of sulfite mutants of Saccharomyces cerevisiae

Sulfite-resistant and sulfite-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. Genetic analysis indicated that one and four genes were responsible for the resistant and sensitive responses, respectively, and suggested that defects in methionine and cysteine metabolism were not involved. Some resistant alleles, all of which were dominant, conferred greater resistance than others. Mutations conferring sensitivity were recessive and one co-segregated with impaired respiration. Two of the sensitive mutants exhibited cross-sensitivity to other metabolic inhibitors: sulfometuron methyl, cycloheximide, oligomycin, and antimycin A. A 50% glutathione deficiency in one sensitive mutant was not sufficient in itself to account for its sensitivity. Screening of other relevant mutants revealed that relative to wild-type, met8 and a thioredoxin null mutant are sensitive, and met3 and met14 mutants are not. Reduced production of extracellular acetaldehyde, a compound that detoxifies sulfite, was observed in three of the four sensitive mutants. However, acetaldehyde was also underproduced in the resistant mutant. Because sulfite is a reducing agent, cells were tested for coincident sensitivity or resistance to ascorbate, selenite, dithiothreitol, nitrite, thiosulfate, reduced glutathione, and cysteine. No consistent pattern of responses to these agents emerged, suggesting that the response to sulfite is not a simple function of redox potential.

Fzf1p of Saccharomyces cerevisiae is a positive regulator of SSU1 transcription and its first zinc finger region is required for DNA binding

The FZF1 gene of Saccharomyces cerevisiae encodes a five‐zinc‐finger transcription factor involved in sulphite tolerance. Previous work based on multicopy suppression analysis placed FZF1 upstream of SSU1, which encodes a plasma membrane protein and putative transporter also implicated in sulphite detoxification. Consistent with this analysis, Fzf1p was found to be a positive regulator of SSU1 transcription. The SSU1 promoter region involved in activation by FZF1 was defined, and the Fzf1 protein was shown to bind to it directly in vitro. Deletion of a single, amino‐terminal zinc finger of Fzf1p resulted in loss of DNA binding, while the fourth and fifth zinc fingers were found to be dispensable. Copyright

Catecholaminergic CATH.a cells express predominantly δ-opioid receptors

Abstract CATH.a cells are a catecholaminergic cell line of neuronal origin. The opioid receptor complement expressed by CATH.a cells was defined pharmacologically and by reverse transcription-polymerase chain reaction (RT-PCR). CATH.a cells were found to express mRNA encoding all three of the major subtypes of opioid receptors. The relative abundance of CATH.a cell opioid receptor transcripts was δ > κ > μ . Pharmacological and functional data were in agreement with the results of RT-PCR inasmuch as δ -opioid receptor was identified as the most abundant opioid receptor subtype expressed by CATH.a cells. In addition, at least one of the opioid signalling pathways, inhibition of adenylyl cyclase activity, was found to be operant in this cell line. CATH.a cells should be of general utility for the study of opioid receptor signalling mechanisms in the context of catecholaminergic neurons.